Hepatocellular carcinoma (HCC) is the 3rd most lethal cancer worldwide. Liver transplant remains the only therapy with a favorable prognosis, and even in these cases, 5-year survival rates are low and recurrence is common 1. Moreover, donor livers are in short supply, and the cost and technical difficulty of transplantation puts therapy out of reach of all by a small minority of people stricken with the disease. Clearly there is a need for pharmaceutical-based therapies for this disease, even if just to extend longevity after transplant. However, the complexities of liver metabolism have kept this goal out of reach. Cytoplasmic thioredoxin reductase (Txnrd1) plays a major role in liver metabolism. Many anti-cancer drugs or drug-candidates, as well as some compounds thought to be active in prophylaxis against cancer initiation, either affect Txnrd1 activity or are substrates for metabolism by Txnrd1 5,6. This suggests that Txnrd1 plays crucial roles in cancer initiation, persistence, and progression. However, prior to now, no studies have been performed in systems having complete and specific genetic disruption of Txnrd1. Therefore, it is unclear what role Txnrd1 plays in responses to these compounds, and what roles undefined 'other targets'might play. We have developed the first animal model with Txnrd1-deficient hepatocytes 3. In the proposed study, we will develop the first animal model with Txnrd1-deficient HCC and to use this model to study initiation, persistence, and progression of HCC in cells that either have or lack Txnrd1. To do this, we have put forth two Specific Aims. First, we will determine whether Txnrd1-deficient hepatocytes are either more susceptible or more refractory than normal hepatocytes to initiation of HCC. Second, we will determine whether Txnrd1 activity is necessary, advantageous, or antagonistic for persistence and progression of HCC cells in situ. This two-year Developmental R21 project is proposed with the intention of establishing and publicly disseminating a novel and powerful animal model for studying HCC, and to provide important understanding of the roles of Txnrd1 in cancer initiation and persistence/progression. PUBLIC HEALTH RELEVANCE: Globally, hepatocellular carcinoma (HCC) is the 3rd most lethal cancer and the most prevalent liver cancer 7. Generally it is a sequelae of chronic inflammatory liver diseases, including hepatitis B and C virus infections, alcoholic cirrhosis, and hepatotoxic exposures 8. The National Cancer Institute (NCI) lists HCC as the most rapidly increasing cancer in the US. HCC causes 14,000 annual deaths in the US and ranks 8th in cancer- related deaths among men. Untreated, HCC is usually fatal within less than a year of primary diagnosis. Since it is often diagnosed at an advanced stage and associated with severe cirrhosis, treatments can be complicated, ineffective, and poorly tolerated, and the prognosis is generally poor (http://pathology2.jhu.edu/ liver/intro.cfm). The preferred treatment is surgical removal and replacement of the cancerous liver with a non- diseased donor-liver (http://www.mayoclinic.com/health/liver-cancer/DS00399/DSECTION=treatments). There are far more people on waiting lists for donor livers than there are livers available. Thus, there is a critical need for improved strategies for preventing, detecting, and treating HCC. HCC does not respond well to current modes of chemotherapy. Whole-liver replacement, the only therapy with a favorable prognosis, has a high incidence of recurrence. Prognostic markers are being refined in hopes of directing transplants to more likely successful patients and efforts are being made to promote earlier diagnosis. However, efforts toward pharmaceutical treatments, perhaps in combination with surgical procedures, should not be abandoned. The combinations of metabolic activities that could be potentially targeted in HCC are vast and relatively un-explored. Continued investigation will eventually lead to improved strategies. Thioredoxin reductase 1 (Txnrd1) has been viewed as a promising cancer drug-target for many years. There at least two dozen drugs or drug-candidates that have Txnrd1-inhibitory activities;another large group of compounds, including potent metal-based anti-cancer drugs, are bio-converted by Txnrd1 into compounds with different physiological activities and toxicities. In addition, we have shown that disruption of Txnrd1 activates drug metabolism pathways, which will likely alter drug activities in hepatocytes. Are there, somewhere in this richly complex metabolic-pharmacologic puzzle, solutions that could help increase the survivability of HCC? We believe there are;however a better understanding of the metabolic and response pathways in liver must be achieved to foster more productive combinatorial trials. To this end, we have developed the first viable animal models entirely lacking hepatocytic Txnrd1, and here we propose to develop the first cancer model, an HCC, lacking Txnrd1. These mice will allow us to far more accurately define the roles of Txnrd1 in cancerous processes and in anti-cancer therapies. In this R21 Grant Application, we propose to develop this HCC model and to use it to resolve the roles of Txnrd1 in initiation and progression of HCC. In the future, we foresee utility for this model in measuring metabolic/pharmacokinetic aspects of potential drug combinations, and in testing efficacy of novel therapeutic strategies on HCC.